Goat anti rabbit hrp secondary antibody

Polyclonal antibody against Ack1 (384) was generated using the GST fusion proteins corresponding to the Pro-rich region (amino acids 502–1008), as described elsewhere [29 (link)]. The monoclonal antibody against Ack1 was generated by Leitat (Barcelona, Spain) and its efficiency has been described previously [18 (link)]. As antigen, we used the same GST fusion protein encoding amino acids 502–1008 of Ack1 as for polyclonal antibodies. Mice were injected with the purified protein, and standard procedures were followed to obtain the antibodies [59 ]. The monoclonal antibody against CAMKII-α was from Affinity Bioreagents (Golden, CO). The goat anti-rabbit-HRP secondary antibody used in the Western Blot analysis was from Sigma (St. Louis, MO), and the antibody against mouse True Blot Ultra WAS from eBioscience (San Diego, CA). Alexa Fluor-488 goat anti-rabbit immunoglobulin G (IgG) (H + L) and Alexa Fluor-546 goat anti-mouse immunoglobulin G (IgG) (H + L) were supplied by Invitrogen (Carlsbad, CA). Protein G-sepharose beads were from Sigma-Aldrich (St. Louis, MO).

To evaluate Esc2 protein expression levels, 10 mL of log-phase cells (OD_600nm ~ 1.0) were harvested and whole cell lysate was extracted using glass bead beating. The concentration of protein in cell lysates were normalized using the Bradford assay (Bio-Rad) and TAF-tagged Esc2 proteins were detected on western blot via chemiluminescence method. The antibodies used were a rabbit anti-Protein A primary antibody (1:10,000, Sigma) and a goat anti-Rabbit HRP secondary antibody (1:10,000).
Protein binding assays were performed using C-terminal 6xHIS tagged Ubc9 was expressed in BL21 cells and purified using a Ni-NTA affinity column. N-terminal Protein-A tagged Esc2 WT and Esc2-D430R mutant proteins were similarly expressed and purified with IgG sepharose resin. 40 μg of Esc2 (WT and D430R) was incubated and bound to 20 μL IgG resin; and was subsequently incubated with 100 μg of purified Ubc9 in a final volume of 200 μL for 2 hours on ice. The IgG resins were then washed 5 times with 1 mL PBS (Phosphate-Bu with 0.2% NP-40. After washing, the bound Ubc9 protein was eluted from the IgG resin with 20 μL of 0.1M glycine-HCl and then 20 μl 1% SDS loading buffer for visualization by Coomassie staining.